Cat. No. / ID: RT34-025
RNase H is an 18.9 kDa recombinant endoribonuclease purified from an Escherichia coli strain, which over-expresses cloned RNase H gene (rnh).
It specifically hydrolyzes the phosphodiester bonds of RNA hybridized to DNA and produces 5´ phosphate-terminated oligoribonucleotides and single-stranded DNA. RNase H does not degrade single and double-stranded DNA or unhybridized RNA. It is a key enzyme in the removal of mRNA after first-strand cDNA synthesis.
It is supplied with 200 mM Tris-HCl (pH 8.4), 500 mM KCl, 50 mM MgCl2 and 200 mM DTT Reaction Buffer (10x).
One unit catalyzes the hydrolysis of 1 nmol of RNA in [3H]-labeled poly(A)×poly(dT) to acid-soluble ribonucleotides in a total reaction volume of 50 µL in 20 minutes at 37°C in 1x Reaction Buffer.
Treating cDNA with RNase H prior to PCR can improve sensitivity as RNA bonded to the cDNA template may prevent binding of the amplification primers in a PCR reaction. RNase H treatment is often necessary when amplifying longer, full-length cDNA targets. In addition, RNase H is useful for removing poly(A) tails on mRNAs after hybridization with oligo(dT) and for the site-specific enzymatic cleavage of RNA.
Protein purity is evaluated using Coomassie Blue detection assay by SDS-PAGE electrophoresis resulting in >90% purity. The relevant procedures have confirmed the absence of DNase, RNase and protease activity.
RNase and DNase contamination is evaluated by assessing RNase activity through gel electrophoresis following the incubation of RNA at 37°C.
Protease activity is also determined by SDS-PAGE electrophoresis.
Use 5 U of the enzyme to remove RNA from an RNA:DNA duplex after reverse transcription in a 20 μL reaction. If a 50 μL reaction is desired, 12.5 U of the enzyme is recommended.
The reaction mixture should be incubated at 37⁰C for 20 minutes.
This is used for applications such as: